Respiratory diseases such as cystic fibrosis (CF) are characterized by a cascade of pathophysiological changes starting with dehydration of mucus secretions and proceeding to impaired mucociliary clearance, chronic airway inflammation, and subsequent lung injury. Effective biomarkers of these changes are urgently needed to monitor disease progression, identify exacerbations, and evaluate the efficacy of existing and novel therapies. The goal of this career development application is to provide Dr. Charles Esther with the skills needed to identify and assess candidate biomarkers in exhaled breath condensate (EBC), an airway sample that can be collected simply and non-invasively even in young children. Dr. Esther plans to overcome the challenges of analyzing the low concentration, variable dilution EBC through application of liquid-chromatography-tandem mass spectrometry (LC-MS/MS). This technology represents a highly sensitive and flexible methodology that permits simultaneous measurement of multiple small molecule biomarkers as well as urea as a dilution marker. In this research proposal, Dr. Esther will use mass spectrometry to identify and test simple and non-invasive EBC biomarkers of two critical aspects of CF airways pathophysiology: neutrophilic airway inflammation and dehydration of airway mucus secretions. Potential biomarkers identified based on both targeted analyses of established biomarkers and metabolomic studies of in vitro models of neutrophilic inflammation and mucus dehydration. Measurement protocols will be developed using LC-MS/MS, and promising biomarkers assessed in a cohort of children and adults with to determine their reliability and relationship to accepted markers of disease pathophysiology and to identify potential confounders. Successful completion of the research plan outlined in this proposal will not only generate important tools for future research on the pathophysiology and therapeutic options for CF, but will also provide Dr. Esther with the skills and experiences necessary to develop a career in identification and testing of non-invasive biomarkers of respiratory disease. RELEVANCE (See instructions): Cystic fibrosis is characterized by disease related changes within the lung, and Dr. Esther proposes to develop simple and non-invasive methods to detect these changes using the cutting edge technologies of mass spectrometry and exhaled breath condensate collection. Development of these methods will provide invaluable tools for clinical management and research in cystic fibrosis and other lung diseases.